1. Field of the Invention
The present invention relates to the fields of PCR and HLA-typing. More specifically, the present invention discloses methods and systems for a tandem PCR process to amplify DNA or RNA within a raw biological specimen and subsequent HLA-typing thereof on an individual or population scale in a field or medical office environment.
2. Description of the Related Art
There is a new and rapidly growing understanding of the medical significance of HLA typing in current medicine. Indeed, there is a very large range of diagnostic and public health applications for HLA-typing. Analysis of the HLA-Locus can be viewed as the historical prototype for the field of genetically personalized therapy (1). DNA-based HLA-Typing has been refined over the past decade into a very accurate companion genetic test for solid organ (2) and bone marrow transplantation therapy (3) and more recently as a companion genetic test for small molecule therapeutics, abacavir (4), lumiracoxib (5) and as genetic screening tests for auto-immune diseases: arthritis (6), celiac disease (7), T1D (8) and a possible screening test for vaccination responsiveness (9). For solid organ or marrow transplantation, high resolution HLA-Typing can be performed via multiple technologies: allele specific PCR (10), Luminex beads (11), Sanger sequencing (12), next generation sequencing (13).
Each of these technologies is accurate and specific enough to support the full range of follow-on HLA-Typing applications. However, the newer HLA-based applications each involve a patient base that is at least 100-times greater than defined by organ transplantation and entail medical treatments which are approximately 100-times less expensive than transplantation. Thus, in order to support the follow-on HLA-based application areas in a resource-limited medical screening environment, the current panel of HLA-Typing technologies must be reduced to tests which can be delivered in the clinic at a test cost in the $5-10 per gene range. Since DNA purification, DNA concentration determination and concentration adjustment comprise a major fraction of the labor and consumable cost associated with HLA-Typing, it would be desirable to develop methods to employ the least expensive of all DNA sources (mouthwash, cheek swabs or saliva) as the sample substrate in a way that bypasses DNA purification, DNA quantitation and DNA concentration adjustment prior to complex genetic testing.
At present, HLA typing requires the effort of an entire molecular genetics laboratory. Incoming blood specimens must first be purified by methods such as spin columns or magnetic beads, followed by quantitation of the purified DNA by methods such as PicoGreen fluorimetry or UV absorbance. The quantified DNA is then subjected to PCR amplification and, following PCR, is then analyzed by high throughput re-sequencing or, more recently, by multiplex hybridization analysis by beads or by microarrays. Thus, the resulting workflow requires the effort of a full molecular genetics laboratory, and at least one full day to compile the final HLA-typing data. The complexity of such a standard workflow also introduces major concerns related to chain-of-custody and the requirement for complex and costly LIMS systems and workflow standard operating procedures, to keep track of sample flow through the several processing and analysis workstations.
Efforts to streamline the process have included obviating DNA purification. Previous attempts to perform PCR amplification from unpurified blood have been problematic even with the availability of variants of the Taq polymerase used for standard PCR. The use of raw blood as a PCR substrate has not yielded consistent results due to the extreme sample-to-sample variation in the white cell complement of blood and possible sample-to-sample variation in the very large excess of blood solutes which can interfere with the underlying PCR reaction.
Mouthwash, cheek swabs and saliva constitute a robust and inexpensive way to collect human DNA for clinical genetics, personalized therapy, and for genetic epidemiology. However, the value of those inexpensive DNA sources is compromised, in part, by the cost and labor required to purify DNA from them, prior to genetic testing.
Thus, there is a recognized need in the art for low equipment and consumable cost, high-throughput methods of gene amplification and HLA typing. There is also a recognized need in the art for developing methods to employ inexpensive DNA sources as the sample substrate in a way that bypasses DNA purification, DNA quantitation and DNA concentration adjustment prior to complex genetic testing. Specifically, the related art is deficient in a hands-free or automated, real-time high-resolution method of HLA typing without a need for first externally purifying the DNA from a sample. The present invention fulfills this long-standing need and desire in the art.